Vaccine against infectious disease

ABSTRACT

The present invention relates to the use of a protein termed 64p in the production of vaccines for protecting animals against the bite of blood-sucking ectoparasites and against the transmission of viruses, bacteria and other pathogens by such ectoparasites.

The present invention relates to the use of a protein termed 64p in theproduction of vaccines for protecting animals against the bite ofblood-sucking ectoparasites and against the transmission of viruses,bacteria and other pathogens by such ectoparasites.

All publications, patents and patent applications cited herein areincorporated in full by reference.

Blood-sucking ectoparasites, such as mosquitoes, horseflies, tsetseflies, fleas, lice, mites and ticks, are extremely effective astransmitters of disease. Mosquito borne diseases include Malaria(Plasmodium parasites transmitted by Anopheles mosquitoes), DengueFever, Yellow Fever and Arboviral Encephalitides (such as Eastern EquineEncephalitis, Japanese Encephalitis, La Crosse Encephalitis, St. LouisEncephalitis (Culex pipiens mosquitoes), Western Equine Encephalitis andWest Nile Virus Encephalitis), Lymphatic filariasis (elephantiasis).Other diseases that are borne by blood-sucking ectoparasite vectorsinclude plague (flea); Schistosomiasis (flatworms); trypanosomiasis(tsetse fly), Leishmaniasis (sandfly), and Onchocerciasis (blackfly).

Taking the tick as an example, these arthropods are able to transmitprotozoan, rickettsial and viral diseases of livestock, which are ofgreat economic importance world-wide. Losses to the livestock industry,in particular the production of cattle and small ruminants in tropicaland sub-tropical areas, have been estimated to be in the range ofseveral billion US dollars annually. In many developing countries,tick-borne protozoan diseases, including Theileria parva which causesthe usually fatal East Coast Fever (Norval et al., 1992a; Norval et al.,1992b), babesioses and rickettsial diseases such as anaplasmoses,cowdriosis and tick-associated dermatophilosis, are major health andmanagement problems of livestock. Furthermore, tick pests also causeconsiderable damage to animals' skin, thereby affecting the leatherindustry. Ticks also act as transmitters of human disease, includingLyme disease (Borrelia burgdorferi, transmitted by Ixodes scapularis),Southern Tick-Associated Rash Illness (STARI), Babesiosis, Ehrlichiosis,Rocky Mountain Spotted Fever (caused by Rickettsia rickettsiatransmitted by Dermacentor variabilis) and Crimean-Congo HaemorrhagicFever.

Normally, these disease agents can only be transmitted by the bite of aninfected ectoparasite. For example, ticks normally become infected bytaking a blood meal from an infected animal. Male, female and immature(nymphs and larvae) ticks feed on blood and all stages are capable oftransmitting disease agents. There are four stages in the life cycle ofa tick: egg, larva, nymph, and adult. It generally takes several monthsto two years to complete this life cycle. A blood meal is taken in allexcept the egg stage. After each blood meal, the cuticle is shed and thetick matures to its next life stage. Thus it is possible for a tick totransmit disease organisms three times in its life. It is also possibleto become infected by handling infected ticks, such as when removingticks from a pet, when infective tick body fluids are introduced into awound or mucus membrane.

In an effort to combat tick-transmitted diseases, a number of attemptshave been made to immunise animals against ticks using extracts of wholeticks or of tick gut. Certain reports have used recombinant tickproteins (see, for example, International patent applicationWO88/03929). WO01/80881 reports the generation of vaccines thatincorporate a protein termed 64P and fragments thereof. However, despitesuch developments, the only commercially-available tick vaccines areactive only against the adult stage of a few tick species and showvariation in efficacy depending on the geographical location of thespecies. No vaccines have yet been developed that provide resistanceacross entire populations of vaccinated animals or against parasites atevery stage of their life cycle.

There is a great need to reduce the incidence of infectious diseasesthat are caused by blood-feeding ectoparasites, particularly in tropicaland sub-tropical regions, where such ectoparasites, and the diseaseagents carried by them, are endemic. A large number of strategies arebeing pursued in order to try and eradicate these diseases, and thesemainly focus on attempting to limit the numbers of the ectoparasitesthemselves. However, to date, none of these strategies has yet shown anyenduring success. Indeed, as the global climate warms, it is likely thatareas not previously afflicted by infectious diseases such as malariawill become vulnerable. The recent outbreak of human encephalitis casesin the north-eastern United States, caused by mosquito-borne West Nilevirus, serves to illustrate the potential problems facing areas that arecurrently temperate over the coming years.

There therefore exists a great need for an effective vaccine to combatdiseases that are transmitted by blood-feeding ectoparasites. It has nowbeen discovered that a protein termed 64p, originally isolated in thetick, is useful as a vaccine component.

SUMMARY OF THE INVENTION

According to the present invention, there is provided a vaccineeffective against the transmission of an infectious disease borne by anectoparasite, said vaccine comprising as an active component a 64pprotein consisting of the sequence presented in FIG. 1, a fragmentthereof or a homologue of said 64p protein or protein fragment thatexhibits at least 50% sequence identity with said protein or proteinfragment.

Immunisation of an animal with such a vaccine is shown herein to causethe generation of antibodies that are effective against a wide varietyof ectoparasite species. The vaccine is also shown to impart protectionagainst the transmission of an infectious disease via a blood-suckingectoparasite.

Although the Applicant does not want to be confined to any particulartheory, it appears that the vaccines of the invention work in thefollowing way. Immunisation of a host species with the 64p protein,fragment or homologue elicits an immune response against theectoparasite protein. This stimulates an inflammatory response thatboosts the immune status of vaccinated animals. However, additionally,the 64p protein and fragments thereof have been discovered to containepitopes that also exist in proteins that are present in the salivaryglands, gut and haemolymph of a large number of ectoparasite species.This cross-reactivity makes the vaccines of the invention particularlyadvantageous, since ingestion of blood, and thus host antibodies, intothe ectoparasite guarantees delivery of the active agent to theparasite. In this manner, the vaccines of the invention target speciesthat feed transitorily, such as mosquitoes and horseflies, asefficiently as those species that remain attached to their host for asignificant period of time, such as ticks.

The nature of the immune response that the vaccines of the inventionimpart is responsible for the decreased transmission of the agent thatcauses the infectious disease borne by the ectoparasite. Because of thepresence in the host of antibodies that recognise not only a proteinspecies present in the ectoparasite saliva, but also a protein speciesthat is found in the gut, the blood-feeding event, however transitory,is sufficient to allow the transmission to the ectoparasite ofantibodies that lead to its death. Immunisation of host animals with avaccine according to the invention thus leads to a decrease in theactual numbers of ectoparasites, as well as a concomitant decrease inthe numbers of ectoparasites carrying disease-causing agents. This has asignificant effect on the incidence of disease per se.

A large number of ectoparasite species exist in various parts of theworld, although their incidence tends to be concentrated in tropical andsub-tropical regions, where they, and diseases carried by them, areendemic. These species vary greatly in type and adopt widely differingfeeding strategies, ranging from transient feeders such as mosquitoes,horseflies, sandflies, blackflies, tsetse flies, fleas, lice and mites,down to flatworms and ticks, some of which may feed for long periods oftime. All of these ectoparasite species are suitable targets for thevaccines of the invention.

What is common between all these ectoparasite species is that theyingest either blood, lymph or they feed on host skin products, meaningthat any antibodies present in their host are automatically internalisedinto the ectoparasite. This provides an advantageous and automatic routeof administration for antibody and, provided that the antibody isreactive against an ectoparasite protein, means that a well-organizedimmunisation regime can result in the complete eradication of theparasite within the area concerned. Ectoparasites that feed on blood areparticularly preferred targets for the vaccines of the invention.

The vaccines of the invention are particularly efficacious against tickspecies. Examples of such targeted tick species are Rhipicephalusappendiculatus, R. sanguineus, R. bursa, Amblyomma variegatum, A.americanum, A. cajennense, A. hebraeum, Boophilus microplus, B.anntulatus, B. decoloratus, Dermacentor reticulatus, D. andersoni, D.marginatus, D. variabilis, Haemaphysalis inermis, Ha. leachii, Ha.punctata, Hyalomma anatolicum anatolicum, Hy. dromedarii, Hy. marginatummarginatum, Ixodes ricinus, I. persulcatus, I. scapularis, I. hexagonus,Argas persicus, A. reflexus, Ornithodoros erraticus, O. moubata moubata,O. m. porcinus, and O. savignyi.

The vaccines of the invention are also particularly efficacious againstmosquito species. Examples of targeted mosquito species are those of theCulex, Anopheles and Aedes genera, particularly Culex quinquefasciatus,Aedes aegypti and Anopheles gambiae.

The vaccines of the invention are also particularly efficacious againstflea species, such as Ctenocephalides felis (the cat flea).

As discussed above, blood-sucking ectoparasites are extremely effectiveas transmitters of infectious disease. Examples of disease agents, thetransmission of which may be prevented or reduced using a vaccineaccording to the present invention include those transmitted bymosquitoes, horseflies, sandflies, blackflies, tsetse flies, fleas,lice, mites, flatworms and ticks. Examples of mosquito-borne diseasesinclude Malaria, Dengue Fever, Yellow Fever and ArboviralEncephalitides, including Eastern Equine Encephalitis, JapaneseEncephalitis, La Crosse Encephalitis, St. Louis Encephalitis, WesternEquine Encephalitis and West Nile Virus Encephalitis and Lymphaticfilariasis. Other diseases whose transmission may be prevented includeplague (flea); schistosomiasis (flatworms); trypanosomiasis (tsetsefly), Leishmaniasis (sandfly), and Onchocerciasis (blackfly). Examplesof tick-transmitted diseases whose transmission may be blocked includeprotozoan, rickettsial and viral diseases of livestock, including EastCoast Fever, babesioses, anaplasmoses, cowdriosis and tick-associateddermatophilosis, and human diseases, such as Lyme's Disease, SouthernTick-Associated Rash Illness (STARI), Babesiosis, Ehrlichiosis, RockyMountain Spotted Fever, tularemia, tick-borne relapsing fever,tick-borne encephalitis (TBE) and Crimean-Congo Haemorrhagic Fever. Theparticular utility of the vaccines of the invention in preventingtransmission of TBE virus has been illustrated herein.

Preferably, the vaccines of the invention are effective againsttransmission of human diseases. However, these vaccines are alsoeffective against the transmission of diseases in mammals (particularlylivestock), birds, reptiles and fish.

By “64p” protein is meant a protein comprising the sequence presented inFIG. 1 herein, or a homologue thereof. This protein and its propertiesare described in detail in co-owned, co-pending International patentapplication WO01/80881, the content of which is incorporated herein inits entirety. This protein contains at least one immunogenic epitopethat is present in all blood-feeding ectoparasites that have been testedso far. It thus appears as if one single vaccine composition may beeffective as a broad spectrum vaccine against all of the ectoparasitespecies that produce a protein containing this common epitope. Theavailability of a single vaccine that is effective against a number ofdifferent ectoparasite species will reduce the cost of administering thevaccine and will thus be advantageous over currently available vaccines.

The sequence in FIG. 1 is the 64p sequence from the tick Rhipicephalusappendiculatus. This protein possesses a sequence typical of astructural protein, and appears to be secreted in the saliva of ticks.The sequence comprising the first 40 amino acids of the cement proteinis strongly collagen-like, whereas the rest of the sequence resembleskeratin. Homology searches conducted with the sequence of this proteinreveals that the highest level of homology for all searched sequences inthe Genbank database (http://www.ncbi.nlm.nih.gov) was 51%, for mouseepidermal keratin subunit I. The protein is glycine-rich and containsseveral repeats of the motif (C/S) 1-4 (Y/F), resembling structuralproteins from Drosophila melanogaster (cuticular protein) and otherinsect egg shells, as well as vertebrate cytokeratins includingmammalian keratin complex 2 basic protein, mouse keratin, human keratin,collagen type IV alpha, and IPIB2 precursor.

The term “homologue” is meant to include reference to paralogues andorthologues of the 64p sequence explicitly identified herein, including,for example, the 64p protein sequence from other tick species, includingR. sanguineus, R. bursa, Amblyomma variegatum, A. americanum, A.cajennense, A. hebraeum, Boophilus microplus, B. annulatus, B.decoloratus, Dermacentor reticulatus, D. andersoni, D. marginatus, D.variabilis, Haemaphysalis inermis, Ha. leachii, Ha. punctata, Hyalommaanatolicum anatolicum, Hy. dromedarii, Hy. marginatum marginatum, Ixodesricinus, I. persulcatus, I. scapularis, I. hexagonus, Argas persicus, A.reflexus, Ornithodoros erraticus, O. moubata moubata, O. m. porcinus,and O. savignyi. The term “homologue” is also meant to include the 64pprotein sequence from mosquito species, including those of the Culex,Anopheles and Aedes genera, particularly Culex quinquefasciatus, Aedesaegypti and Anopheles gambiae; flea species, such as Ctenocephalidesfelis (the cat flea); horseflies; sandflies; blackflies; tsetse flies;fleas; lice; mites; leeches; and flatworms.

Methods for the identification of homologues of the 64p protein will beclear to those of skill in the art. For example, homologues may beidentified by homology searching of sequence databases, both public andprivate. Conveniently, publicly available databases may be used,although private or commercially-available databases will be equallyuseful, particularly if they contain data not represented in the publicdatabases. Primary databases are the sites of primary nucleotide oramino acid sequence data deposit and may be publicly or commerciallyavailable. Examples of publicly-available primary databases include theGenBank database (http://www.ncbi.nlm.nih.gov/), the EMBL database(http://www.ebi.ac.uk/), the DDBJ database (http://www.ddbj.nig.ac.jp/),the SWISS-PROT protein database (http://expasy.hcuge.ch/), PIR(http://pir.georgetown.edu/), TrEMBL (http://www.ebi.ac.uk/), the TIGRdatabases (see http://www.tigr.org/tdb/index.html), the NRL-3D database(http://www.nbrfa.georgetown.edu), the Protein Data Base(http://www.rcsb.org/pdb), the NRDB database(ftp://ncbi.nlm.nih.gov/pub/nrdb/README), the OWL database(http://www.biochem.ucl.ac.uk/bsm/dbbrowser/OWL/) and the secondarydatabases PROSITE (http://expasy.hcuge.ch/sprot/prosite.html), PRINTS(http://iupab.leeds.ac.uk/bmb5dp/prints.html), Profiles(http://ulrec3.unil.ch/software/PFSCAN_form.html), Pfam(http://www.sanger.ac.uk/software/pfam), Identify(http://dna.stanford.edu/identify/) and Blocks(http://www.blocks.fhcrc.org) databases. Examples ofcommercially-available databases or private databases includePathoGenome (Genome Therapeutics Inc.) and PathoSeq (IncytePharmaceuticals Inc.).

Typically, greater than 30% identity between two polypeptides(preferably, over a specified region) is considered to be an indicationof functional equivalence and thus an indication that two proteins arehomologous. Preferably, proteins that are homologous to the 64p proteinhave a degree of sequence identity with the 64p protein, or with activefragments thereof, of greater than 55%. More preferred homologues havedegrees of identity of greater than 40%, 50%, 60%, 70%, 80%, 90%, 95%,98% or 99%, respectively with the 64p protein, or with active fragmentsthereof. Percentage identity, as referred to herein, is as determinedusing BLAST version 2.1.3 using the default parameters specified by theNCBI (the National Center for Biotechnology Information;http://www.ncbi.nlm.nih.gov/) [Blosum 62 matrix; gap open penalty=11 andgap extension penalty=1].

Homologues of the 64p protein include mutants containing amino acidsubstitutions, insertions or deletions from the wild type sequence,provided that the immunogenicity of the wild type protein sequence isretained. Mutants thus include proteins containing conservative aminoacid substitutions that do not affect the function or activity of theprotein in an adverse manner. This term is also intended to includenatural biological variants (e.g. allelic variants or geographicalvariations within the species from which the tissue cement proteins arederived). Mutants with improved immunogenicity from that of the wildtype protein sequence may also be designed through the systematic ordirected mutation of specific residues in the protein sequence.

For the avoidance of doubt, also embraced by the term “homologues” arethose proteins whose encoding genes have not yet been currently cloned,but which are cloned in the future. Methods for cloning genes that arehomologues of the proteins of the invention will be known to those ofskill in the art. For example, a nucleic acid molecule from the geneencoding 64p from R. appendiculatus (see FIG. 1) as described above maybe used as a hybridization probe for RNA, cDNA or genomic DNA isolatedfrom an ectoparasite, in order to isolate full-length cDNAs and genomicclones encoding the equivalent 64p protein in this species. In thisregard, the following techniques, among others known in the art, may beutilised and are discussed below for purposes of illustration.

One such method is to probe a genomic or cDNA library with a natural orartificially-designed probe using standard procedures that arerecognised in the art (see, for example, “Current Protocols in MolecularBiology”, Ausubel et al. (eds). Greene Publishing Association and JohnWiley Interscience, New York, 1989,1992). Probes comprising at least 15,preferably at least 30, and more preferably at least 50, contiguousbases that correspond to, or are complementary to, nucleic acidsequences from an appropriate encoding gene, such as that set out inFIG. 1 herein.

Such probes may be labelled with an analytically-detectable reagent tofacilitate their identification. Useful reagents include, but are notlimited to, radioisotopes, fluorescent dyes and enzymes that are capableof catalysing the formation of a detectable product. Using these probes,the ordinarily skilled artisan will be capable of isolatingcomplementary copies of genomic DNA, cDNA or RNA polynucleotidesencoding proteins of interest from human, mammalian or other animalsources and screening such sources for related sequences, for example,for additional members of the family, type and/or subtype.

In many cases, isolated cDNA sequences will be incomplete, in that theregion encoding the polypeptide will be cut short, normally at the 5′end. Several methods are available to obtain full length cDNAs, or toextend short cDNAs. Such sequences may be extended utilising a partialnucleotide sequence and employing various methods known in the art todetect upstream sequences such as promoters and regulatory elements. Forexample, one method which may be employed is based on the method ofRapid Amplification of cDNA Ends (RACE; see, for example, Frohman etal., Proc. Natl. Acad. Sci. USA (1988) 85: 8998-9002). Recentmodifications of this technique, exemplified by the Marathon™ technology(Clontech Laboratories Inc.), for example, have significantly simplifiedthe search for longer cDNAs. A slightly different technique, termed“restriction-site” PCR, uses universal primers to retrieve unknownnucleic acid sequence adjacent a known locus (Sarkar, G. (1993) PCRMethods Applic. 2:318-322). Inverse PCR may also be used to amplify orto extend sequences using divergent. primers based on a known region(Triglia, T., et al. (1988) Nucleic Acids Res. 16:8186). Another methodwhich may be used is capture PCR which involves PCR amplification of DNAfragments adjacent a known sequence in human and yeast artificialchromosome DNA (Lagerstrom, M. et al. (1991) PCR Methods Applic. 1:111-119). Another method which may be used to retrieve unknown sequencesis that of Parker, J. D. et al. (1991); Nucleic Acids Res.19:3055-3060). Additionally, one may use PCR, nested primers, andPromoterFinder™ libraries to walk genomic DNA (Clontech, Palo Alto,Calif.). This process avoids the need to screen libraries and is usefulin finding intron/exon junctions.

When screening for full-length cDNAs, it is preferable to use librariesthat have been size-selected to include larger cDNAs. Also,random-primed libraries are preferable, in that they will contain moresequences that contain the 5′ regions of genes. Use of a randomly primedlibrary may be especially preferable for situations in which an oligod(T) library does not yield a full-length cDNA. Genomic libraries may beuseful for extension of sequence into 5′ non-transcribed regulatoryregions.

Methods for DNA sequencing and analysis are of course well known and aregenerally available in the art. Preferably, the sequencing process maybe automated using machines such as the Hamilton Micro Lab 2200(Hamilton, Reno, Nev.), the Peltier Thermal Cycler (PTC200; MJ Research,Watertown, Mass.) and the ABI Catalyst and 373 and 377 DNA Sequencers(Perkin Elmer).

Fragments of the 64p protein are also useful as components of thevaccines of the present invention. Included as such fragments are notonly fragments of the Rhipicephalus appendiculatus 64p protein that isexplicitly identified herein in FIG. 1, but also fragments of homologuesof this protein. Such homologous fragments will typically possessgreater than 30% identity with the R. appendiculatus 64p sequence,although more preferred homologues will display degrees of identity ofgreater than 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99%, respectivelywith the 64p protein fragments that are explicitly identified herein.

For example, short stretches of peptide derived from immunogenicportions of 64p proteins may be particularly useful as immunogens. Suchshort stretches of polypeptide sequence are simple to produce in largequantities, either synthetically or through recombinant means. Proteinfragments may in many instances be preferred for use in the vaccines ofthe invention, since these fragments are likely to fold intoconformations not adopted by the full length wild type 64p sequence.Since some 64p proteins are likely to have evolved so as to resemble thetissues of the host skin and thus to avoid provoking a host immuneresponse against the tick, such unnatural forms of tick cement proteinsthat expose ‘hidden’ epitopes are likely to be of particular use in thevaccines of the present invention.

Examples of fragments of 64p proteins that are useful for inclusion inthe vaccine compositions of the invention include various fragments thathave been generated recombinantly by the inventors (see WO01/80881), andhomologues of these fragments and mutants of the kind discussed above.As will be apparent to the skilled reader, similar fragments to thosethat are explicitly disclosed herein may be prepared from ectoparasitespecies other than ticks.

The details of the R. appendiculatus fragments described herein are asfollows.

The fragment termed 64trp1 is a small soluble C-terminal fragment of the64P protein consisting of 29 amino acids (residues 103-132 inclusive ofthe sequence of FIG. 1) cloned as a glutathione-S-transferase(GST)/histidine tag fusion protein with a molecular weight of around 30kDa.

The fragment termed 64trp2 refers to a small soluble N-terminal fragmentof the 64P protein consisting of 51 amino acids (residues 1-51 inclusiveof the sequence of FIG. 1) cloned as a glutathione-S-transferase(GST)/histidine tag fusion protein with a molecular weight of around 33kDa.

The fragment termed 64trp3 refers to a larger soluble N-terminalfragment of 64P protein consisting of 70 amino acids (residues 1-70inclusive of the sequence of FIG. 1) cloned as aglutathione-S-transferase (GST)/histidine tag fusion protein with amolecular weight of around 36 kDa.

The fragment termed 64trp4 is a soluble C-terminal fragment of 64Pprotein consisting of 63 amino acids (residues 69-132 inclusive of thesequence of FIG. 1) cloned as a glutathione-S-transferase(GST)/histidine tag fusion protein with a molecular weight of around 35kDa.

The fragment termed 64trp5 is the full-length clone of 64P proteinsequence consisting of 133 amino acids cloned as a GST fusion protein(i.e. minus the histidine tag). This protein is soluble and has amolecular weight of 41 kDa.

The fragment termed 64trp6 refers to the full-length clone of 64Pprotein consisting of 133 amino acids cloned as aglutathione-S-transferase (GST)/histidine tag fusion protein. Thisfragment is insoluble and has an approximate molecular weight of around42 kDa.

These protein fragments, and homologues thereof, are particularlypreferred components for incorporation in the vaccines of the invention.These fragments may be expressed as soluble protein, or mayalternatively be expressed in inclusion bodies and purified underdenaturing conditions. For example, the construct 64trp6 as isolatedfrom R. appendiculatus has been prepared as a denatured proteinexpressed in inclusion bodies and demonstrated to be immunogenic in thisform.

Immunisation with these protein fragments, followed by attachment ofectoparasite, results in inflammation at the attachment site andsubsequent death of the ectoparasite. The skilled reader will appreciatethat the presence of the heterologous GST and HIS tag sequences ispurely for convenience of protein production. These stretches ofsequence are not considered to be essential to this aspect of theinvention.

Conveniently, the vaccines according to the invention contain a 64pprotein, fragment thereof or homologue thereof, expressed in recombinantform. Recombinantly-expressed protein is inexpensive to produce and,using the now standard techniques of genetic engineering, allows thesimple manipulation of gene sequences to give a desired protein product.

It is preferred that the vaccines of the invention are effective againstboth adult and immature forms of the ectoparasite. The term “immature”is meant to include both nymph and larval forms of the ectoparasite.This means that the whole ectoparasite population may be targeted usingthe vaccine, so increasing the efficiency of eradication of ectoparasiteand infectious disease causing agent.

The vaccines may specifically target adult or immature forms ofectoparasites, but will preferably target all parasitic stages of thelife cycle. Of the fragments specifically exemplified herein, 64trp2-,64trp3-, 64trp5- and 64trp6-immunised animals has been found to causesignificant mortality in tick nymphs or adult ticks or both nymphs andadults, depending on the tick species, and these fragments are thusparticularly preferred.

According to a further embodiment of the invention, there is provided acocktail vaccine comprising, in addition to the 64p protein, fragment orhomologue, a second active agent. The second active agent may preferablybe a second immunogenic protein, or protein fragment derived from ablood-feeding ectoparasite. More preferably, the second immunogenicprotein, fragment or homologue is a 64p protein or protein fragment.

The second active agent may be a vaccine against an infectious disease.It has been found that whilst certain commercially available vaccinesagainst infectious disease are effective in abrogating the effects onthe host of the virus that causes the infectious disease and thus inprotecting against lethal challenge with virus-infected ticks, thesevaccines do not protect the host against infection. This is shown herein(see Example 3.10) by the ability of immunised hosts to support virustransmission to uninfected ticks feeding upon them.

The cocktail vaccine produced by the combination of a 64p-based vaccineaccording to the invention and a vaccine against an infectious diseasewould be effective in preventing viral transmission through theproperties of the 64p components described above, and would also protectagainst lethal viral challenge through the properties of the vaccineagainst infectious disease. Such a combined vaccine should thus protectagainst both host infection and death.

Preferably, the vaccine against an infectious disease used as the secondagent is a vaccine against TBE. Examples of commercially-available TBEvaccines are known to those of skill in the art.

Optionally, the cocktail vaccine may contain an adjuvant.

For example, any two or more immunogenic 64p proteins, protein fragmentsor functional equivalents may be used as components of such a cocktailvaccine, and may be from different or from the same tick species. Forexample, it may be desired to generate a vaccine that specificallytargets more than one ectoparasite, or that targets different proteinsfrom the same ectoparasite. In this manner, it may be possible togenerate a more efficacious vaccine with greater species coverage.Particularly preferred combinations of components include thecombination of 64trp2, 64trp3, 64trp5 and 64trp6, the combination of64trp2 and 64trp5, the combination of 64trp2 and 64trp6 and thecombination of 64trp5 and 64trp6. These combinations are demonstratedherein to possess particular efficacy in targeting both adult andimmature ticks, in conferring cross-species resistance and in blockingthe transmission to the host of the disease-causing agent.

Vaccine compositions according to the invention may also compriseadditional agents, for example, molecules that the ectoparasite uses topromote pathogen transmission, such as interferon regulators, complementinhibitors, chemokine regulators and immunoglobulin-binding proteins. Inthis way, other bioactive molecules that are released from the salivaryglands of ectoparasites may be recognised as foreign by the host immunesystem and an immune response mounted.

A further aspect of the present invention comprises a vaccine containinga 64p protein, fragment or homologue fused to another molecule, such asa label, a toxin or other bioactive or immunogenic molecule.Particularly suitable candidates for fusion may be a molecule such asglutathione-S-transferase or a histidine tag, although luciferase, greenfluorescent protein or horse radish peroxidase may also be suitable.Linker molecules such as streptavidin or biotin may also be used, forexample, to facilitate purification of the cement protein.

Fusion proteins may be created chemically, using methods such aschemical cross-linking. Such methods will be well known to those ofskill in the art and may comprise, for example, cross-linking of thethiol groups of cysteine residues. Chemical cross-linking will in mostinstances be used to fuse tissue cement proteins to non-proteinmolecules, such as labels.

When it is desired to fuse a tissue cement protein to another proteinmolecule, the method of choice will generally be to fuse the moleculesgenetically. In order to generate a recombinant fusion protein, thegenes or gene portions that encode the proteins or protein fragments ofinterest are engineered so as to form one contiguous gene arranged sothat the codons of the two gene sequences are transcribed in frame.

Immunisation with naked, plasmid DNA encoding specific antigens hasrecently been acknowledged as an efficient method of presenting antigensto the mammalian immune system, resulting in strong humoral and cellularimmune responses (Ulmer et al., Science 1993, 259, 1745-1749). Thistechnique, also referred to as DNA vaccination, has been successfullyapplied to generate antibodies directed against several proteins derivedfrom viruses (Ulmer et al., loc cit.; Cox et al., J. Virol. 1993, 67,5664-5667; Fynan et al., Proc. Natl. Acad. Sci. USA 1993, 90,11478-11482; Robinson et al., Vaccine 1993, 11, 957-960; Wang et al.,1993, DNA Cell Biol. 1993, 12, 799-805; Davis et al., Hum. Mol. Genet.1993, 2, 1847-1851; Xiang et al., Virology 1994, 199, 132-140; Xiang etal., Virology 1995, 209, 569-579; and Justewicz et al., J. Virol. 1995,69, 7712-7717), parasites (Sedegah et al., Proc. Natl. Acad. Sci. USA1994, 91, 9866-9870; Mor et al., J. Immunol. 1995, 155, 2039-2046; andYang et al., Biochem. Bioph. Res. Comm. 1995, 212, 1029-1039) andbacteria (Anderson et al., Infect. Immun. 1996, 64, 3168-3173), and, inseveral cases, a significant protective response has been elicited bythe host. These DNA vaccines continuously stimulate the immune system,amplifying immunity and thereby reducing the cost of production anddelivery as no booster injections are required.

Based on the available evidence, immunisation with plasmid DNA encodingthe various 64p proteins is likely to be a useful technique to improvetheir anti-tick vaccine effects further. The method would involve directinjection of the host with a eukaryotic expression vector such that oneor more 64p proteins are expressed by in vivo transcription thentranslation of the corresponding sequence within the vaccinated host(humans, livestock, or other animals).

The vaccines of any one of the above-described aspects of the inventionmay additionally comprise an adjuvant. Suitable adjuvants to enhance theeffectiveness of the immunogenic proteins according to the presentinvention include, but are not limited to, oil-in-water emulsionformulations (optionally including other specific immunostimulatingagents such as muramyl peptides or bacterial cell wall components), suchas for example (a) those formulations described in PCT Publ. No. WO90/14837. Other suitable adjuvants will be known to those of skill inthe art and include Saponin adjuvants, such as TiterMax Gold (CytRxCorporation, 150 Technology Parkway, Atlanta Norcross, Ga.), Stimulon™(Cambridge Bioscience, Worcester, Mass.), ISA Montanide 50, cytokines,such as interleukins, interferons, macrophage colony stimulating factor(M-CSF) or tumor necrosis factor (TNF).

According to a further embodiment of the invention; there is provided amonoclonal antibody that is reactive with a 64p protein and which isthus effective in blocking the transmission of a disease-causing agent.By “reactive” is meant that the antibody binds to one or more epitopesof a 64p protein with an affinity of at least 10⁻⁸M, preferably at least10⁻⁹M, more preferably at least 10⁻¹⁰M. According to a preferredembodiment of this aspect of the invention, the antibody or antiserum isreactive against analogues of the 64p protein, for example, 64p proteinanalogues from a number of different ectoparasite species, such as thetick, mosquito, sandfly, blackfly and so on. This aspect of theinvention includes a method for the production of such an antibody or anantiserum, comprising immunising an animal with a 64p protein, fragmentthereof, or homologue thereof as listed in any one of theabove-described aspects of the invention.

According to a still further aspect of the invention, there is provideda process for the formulation of a vaccine composition comprisingbringing a 64p protein, fragment or homologue into association with apharmaceutically-acceptable carrier, optionally in conjunction with anadjuvant. The technology referred to as jet injection (see, for example,www.powderject.com) may also be useful in the formulation of vaccinecompositions.

According to a still further aspect of the present invention, there isprovided a method of immunising a mammal against anectoparasite-transmitted disease or against a blood-feedingectoparasite, comprising administering to an animal, a vaccine accordingto any one of the above-described aspects of the invention. Such animmunisation method may utilise conventional means, but alternativemethods of administering vaccines, such as through the use of jetinjection may be equally effective or even preferable (see, for example,www.powderject.com; also Sarno et al. (2000) Pediatr. Infect. Dis. J.19:839-842).

The invention also provides a 64p protein, fragment thereof or homologuethereof, for use in a vaccine. The invention further provides for theuse of a 64p protein, fragment thereof or homologue thereof as acomponent of a vaccine.

Various aspects and embodiments of the present invention will now bedescribed in more detail by way of example, with particular reference tothe 64p protein from the tick, Rhipicephalus appendiculatus. It will beappreciated that modification of detail may be made without departingfrom the scope of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Nucleotide and inferred amino acid sequences of 64p: CompletecDNA sequence and cDNA-inferred protein sequence of clone 64. Theputative signal sequence is given in bold. A possible glycosaminoglycanattachment site is underlined. The first 40 amino-acid piece of themature protein is collagen-like, the remainder of the sequence resembleskeratin. The protein is glycine-rich and contains several repeats of themotif (C/S)1-4(Y/F), which is also found in structural proteins frominsect egg shells. The tyrosines may be involved in cross-linking byformation of dityrosine-bridges by phenyloxidases. * indicates the stopcodon.

FIG. 2: Amino acid sequences of 64P protein fragments (64TRPs) expressedin Escherichia coli. P1/P2, P1/P3, P4/P5, P6/P5, P1/P5 and P7/P5 referto primers used to subclone PCR products from 64P amino acid sequenceinto the plasmid pGEX-2T, for expression in Escherichia coli cells astruncated versions of 64P protein, i.e. 64trp2 (51 amino acids), 64trp3(70 amino acids), 64trp1 (29 amino acids), 64trp4 (63 amino acids),64trp5 (133 amino acids without HIS.TAG) and 64trp6 (133 amino acidswith HIS.TAG), respectively. Predicted possible cleavage signal peptide(amino acids 1 to 18) is underlined in green.

FIG. 3: Histological studies of skin sections from hamsters immunisedwith different cocktails of 64trp proteins, post-feeding of Ixodesricinus nymphs, stained with Haematoxylin and Eosin. (A)=histologicalsection from skin of GST-immunised, control hamster & (B), (C) &(D)=histological sections from skin of hamsters immunised with 64trpproteins, post-feeding with I. Ricinus nymphs, stained with haematoxylin& eosin stains; sections (A), (B) & (D)—magnification=20×; section(C)—magnification=40×; arrows:1=epidermis, 2=dermis, 3=subcutis &4=cement cone; d=dentritic-like cells (i.e. Langerhan cells on cementcone), f=fibroblasts, l=lymphocytes & m=mast cells.

FIG. 4: Immunoblots of different mosquito antigens probed with (A)anti-64trp2, (B) anti-64trp5, (C) anti-64trp6 and (D) anti-GST(control)sera, respectively; (E) Coomassie Blue stained 4-12% gradient gelshowing the protein profile of the same mosquito antigens.

Lanes 2, 3, 4, 5 & 6 in all figures denote: Lane 2, salivary glandextract of Anopheles gambiae, Lane 3 midgut extract of Anophelesgambiae, Lane 4 midgut extract of Aedes aegypti, Lane 5 midgut extractof Culex quinquefasciatus and Lane 6 salivary gland extract of Culexquinquefasciatus, mosquitoes. Immunopositive bands labelled as a-n;faintly visible bands 1-n refer to non-specific binding of proteins inthe mosquito tissue extracts by the anti-GST control serum.

Lane 1 figures A, B, C & D: SeeBlue™ Plus2 protein molecular weightmarkers: 188 kD=Myosin, 98 kD=Phosphorylase B, 62 kD=Bovine serumalbumin, 49 kD=Glutamic dehydrogenase, 38 kD=Alcohol dehydrogenase, 28kD=Carbonic anhydrase, 17 kD=Myoglobin Red and 14 kD=Lysozyme; lane 1figure E: Mark 12 protein markers: 200 kD=Myosin, 116.3kD=B-galactosidase, 97.4 kD=Phosphorylase b, 66.3 kD=Bovine serumalbumin, 55.4 kD=Glutamic dehydrogenase, 36.5 kD=Lactate dehydrogenase,31 kD=Carbonic anhydrase, 21.5 kD=Trypsin inhibitor, 14.4 kD=Lysozymeand 6 kD=Aprotinin.

FIG. 5: Cross-reactivity between Ctenocephalides felis (cat flea)antigens using antisera from guinea pigs immunised with recombinant R.appendiculatus 64trp proteins. A, B, C, D & E immunoblots of whole fleaextracts of Ctenocephalides felis fed on cats using GST (A), 64trp2 (B),64trp3 (C), 64trp5 (D) and 64trp6 (E) antisera, respectively; FCoomassie Blue stained 4-12% Bis-Tris gradient gel (NuPAGE-Novex) of thesame extracts.

Lanes: A/1, B/1, C/1, D/1 & E/1: See Blue™ Plus 2 protein molecularweight markers (Novex): 98 kD=Phosphorylase b, 62 kD=BSA, 49kD=Glutamine dehydrogenase, 38 kD=Alcohol dehydrogenase, 28 kD=Carbonicanhydrase, 17 kD=Myoglobin Red & 14 kD=Lysozyme. Lane: F/1: Mark 12™protein moleculat weight markers 9Novex): 200 kD=Myosin, 116.3kD=B-galactosidase, 97.4 kD=Phosphorylase b, 66.3 kD=BSA, 55.4kD=Glutamic dehydrogenase, 36.5 kD=Lactate dehydrogenase, 31 kD=Carbonicanhydrase & 21.5 kD=Trypsin inhibitor.

Lanes: B/2, C/2, D/2 & E/2=C. felis whole extract of whichimmuno-positive bands were observed as b=110 kD, c=62 kD, d=75 kD, e=98kD, f=75 kD, g=⁴⁸-50 kD, h=28-31 kD, i=98-120 kD and j=50 kD,respectively. Lane: A/2=C. felis whole extract showing a faintimmuno-positive band observed as a, due cross-reactivity of fleaantigens with anti-GST antiserum.

FIGS. 6-8: Effect of 64TRP constructs in blocking TBE virus transmissionin mice.

EXAMPLES

Expression of truncated cement proteins (64TRP) in bacteria is reportedin WO01/80881, the content of which is incorporated herein in itsentirety.

This patent application also details the expression of the various 64TRPconstructs, along with immunohistochemical studies using antiserum to64TRP, and vaccination trials in Dunkin-Hartley Guinea pigs. It was alsoreported in this application that antisera raised against Rhipicephalusappendiculatus cement protein 64TRP were cross-reactive with antigenicepitopes in the salivary gland, midgut and haemolymph of adult female R.appendiculatus and with antigenic epitopes in the salivary glands,midgut and haemolymph of three other ixodid tick species. The resultssuggested that the candidate vaccine(s) derived from R. appendiculatuscement provide a broad spectrum vaccine that is effective against anumber of different tick species.

On the basis of the observed cross-reactivities, 64trp6 of R.appendiculatus was selected as an immunogen for a vaccine trial. Theresults presented in this specification showed that putative vaccinesderived against cement protein 64TRP of R. appendiculatus werecross-protective against adults and nymphs of R. sanguilzeus.

Following on from this work, additional work has now been performed thathas revealed surprising findings relating to the cross-reactivity of64TRP-based vaccines in insects, and the ability of these vaccines toconfer resistance to infection.

Example 1 Cross-Reactivity and Cross-Protection Vaccine Trial withInsects

1.1 Selection of Immunogens

Candidate immunogens were identified on the basis of whether antiserumto the construct detected specific cross-reacting antigens in extractsof mosquitoes and fleas.

Cross-reactivity studies using immunoblotting with 64trp antisera showeddetection of protein bands with mosquito extracts of Anopheles gambiaesalivary gland and midgut, Aedes aegypti midgut, and Culexquinquefasciatus salivary gland and midgut (FIG. 3).

Using 64trp2 antiserum (FIG. 4A), two major bands (a and b) weredetected in all extracts.

Antisera to 64 trp5 (FIG. 4B) detected several less pronounced bands inmidgut of An. gambiae and C. quinquefasciatus. Antiserum to 64trp6detected a prominent band in An. gambiae midgut (FIG. 4C).

The control antiserum raised against GST detected very faint bands inmidgut of An. gambiae and C. quinquefasciatus that differed in size fromthose detected with 64trp antisera (FIG. 4D).

Based on the availability of A. aegypti larvae, and the observedcross-reactivities, a vaccine trial was undertaken using 64trp2 and 5.

1.2 Treatments for Vaccine Trial:

-   -   Group 1: Recombinant 64trp2+64 trp5+Montanide ISA (2 mice)    -   Group 2: Recombinant 64trp5+Montanide ISA (2 mice)    -   Group 3: Recombinant 64trp2+Montanide ISA (2 mice)    -   Group 4: GST (control) (2 mice)    -   Group 5: Recombinant 64trp2+64 trp5+Montanide ISA (2 guinea        pigs)    -   Group 6: Recombinant 64trp2+Montanide ISA (2 guinea pigs)    -   Group 7: Recombinant 64trp5+Montanide ISA (2 guinea pigs)    -   Group 8: GST (control) (2 guinea pigs)        Total Number of Animals=8 mice+8 guinea pigs        1.3 Route and Dose:

Subcutaneous inoculation in the prescapular region either singly or ascombined antigens into a single site.

Dose 50 μg antigen per guinea pig and 10 μg antigen per mouse.

1.4 Vaccination Scheme:

-   1. Primer inoculation-   2. First boost-   3. Test bleed at 10 to 12 days post-inoculation-   4. Second boost (if antibody titre <{fraction (1/5000)})-   5. Test bleed at 10 to 12 days post-inoculation-   6. Antibody titre >{fraction (1/5000)}: challenge with Aedes aegypti    mosquitoes.-   7. Evaluate local inflammatory immune response to repeated mosquito    feeding, and survival of fed mosquitoes.    1.5 Results

The results are summarised in Tables 1a and 1b. Overall, they show thatputative vaccines derived against cement of R. appendiculatus werecross-reactive against A. aegypti mosquitoes.

(i) Feeding Success

Adult female mosquitoes fed preferentially on young (5-6 week-old) micecompared with guinea pigs.

(ii) Inflammatory Response

Local skin hypersensitivity reactions, observed as intense papularswellings, were observed on the abdomen and foot pads of mice by thethird feeding.

(iii) Post-Feeding mortality

Higher mortality was observed among mosquitoes exposed to64trp-immunised animals compared to control animals.

(iv) Antibody Titres

Antibody titres for Balb/c mice immunised with 64trp2 were >1:64,000 andfor C57/BI10 mice immunised with 64trp2, 1:64,000.

1.6 Conclusions

-   1. Antibodies raised against the 64trp proteins cross-react in    immunoblots with antigenic epitopes in salivary gland and midguts of    adult mosquitoes.-   2. A host inflammatory response was observed in mice immunised with    64trp immunogens.

3. The mortality in mosquitoes fed on 64trp-immunised animals indicatesthat the tick cement protein is a candidate for developing anti-mosquitovaccines. TABLE 1a Effect of feeding Aedes aegypti mosquitoes on miceimmunised with recombinant 64trp proteins Live Dead Total Mouse 64trpmosquitoes mosquitoes MM + Mortality strain protein MM FF MM FF FF (%)FF Balb/C 64trp2/5 19 9 0  5* 33 20.8 Balb/C 64trp5 14 13 0 8 35 30.1Balb/C 64trp2 6 10 0  7* 23 41.2 Balb/C GST 19 16 0 0 35 0 (control)C57/BI10 64trp2/5 10 15 1  6* 33 28.6 C57/BI10 64trp5 8 14 0 2 31 12.5C57/BI10 64trp2 23 9 0 3 35 25 C57/BI10 GST 22 7 0 1 30 12.5 (control)

TABLE 1b Effect of feeding Aedes aegypti mosquitoes on guinea pigsimmunised with recombinant 64trp proteins Guinea Live Dead Mor- pig64trp mosquitoes mosquitoes Total tality no. protein MM FF MM FF MM + FF(%) FF 1a 64trp2/5 19 21 0 8 48 27.6 1b 64trp2/5 26 19 1 6 52 24 2a64trp2 19 14 0 2 35 12.5 2b 64trp2 14 18 0 9 41 33.3 3a 64trp5 16 21 113 51 38.2 3b 64trp5 16 11 0 6 33 35.3 4a GST 18 10 1 0 29 0 (control)4b GST 19 9 0 1 26 10 (control)*female mosquitoes observed dying during counting

Example 2 Antigenic Cross-Reactivity Between Rhipicephalusappendiculatus and Ctenocephalides felis Cat Flea Detected byImmunoblotting Using Antisera to R. appendiculatus Cement Protein 64 trpConstructs

Antisera to 64trp 2, 64trp5, and 64trp6 showed strong cross-reactivitiesin immunoblots of whole cat flea extract probed with the respectiveantisera (FIG. 5). The results are summarised in Table 2 below. Singlecross-reactions were also detected with anti-64trp3 and anti-GST sera.The cross-reactivities demonstrate the potential for developing ananti-flea vaccine using the tick cement protein. TABLE 2Cross-reactivity between Rhipicephalus appendiculatus andCtenocephalides felis whole flea extract using sera from guinea pigsimmunised with 64 trp recombinant antigens Tick Antigens R.appendiculatus Antiserum CC SG gut H N L C. felis Anti-64trp2ab′ + + + + + + + (50a.a. N-term. Frag. of 64P) Effective against RAAdult/nymph ticks (soluble antigen) Anti-64trp3 ab′ + + + + +− + +(70a.a. N-term. Frag. of 64P) Effective against RA Adult/nymphs ticks(soluble antigen) Anti-64trp5 ab′ + + + + − − + (133a.a. full-lengthclone of 64P) effective against RA nymph ticks (soluble antigen)Anti-64trp6 ab′ + + + + + + + (133a.a. full-length clone of 64P)effective against RA nymph ticks (de- natured antigen) Anti-GST ab′control − + − − − − + antiserumCC = tick cement cone extract;SG = salivary gland extract,gut = midgut extract;H = haemolymph;N = whole nymphal extract;L = whole larval extract;+ = positive and− = negative reactions, respectively, to antisera used in immunoblots;ab′ = antiserum

Example 3 Evaluation of 64p Anti-Tick Vaccine Constructs for TheirAbility to Protect Mice Against Tick-Borne Encephalitis (TBE) VirusInfection

3.1 Selection of Immunogens

Candidate immunogens were selected on the basis of whether antiserum tothe 64TRP constructs detected specific cross-reacting antigens inextracts of Ixodes ricinus, the tick vector of TBE virus (see Table 3 ofWO01/80881).

3.2 Treatments for Vaccine Trial (Trial 1):

-   -   Group A: Recombinant 64trp2+TiterMax Gold (TMG) (10 mice)    -   Group B: Recombinant 64trp5+TMG (10 mice)    -   Group C: Recombinant 64trp2+64trp6+TMG (10 mice)    -   Group D: Recombinant 64trp5+64trp6+TMG (10 mice)    -   Group E: Recombinant 64trp2+64trp5+TMG (10 mice)    -   Group F: GST protein (10 mice)    -   Group G: TMG (10 mice)    -   Group H: untreated (10 mice)        Total number of animals=80 Balb/c mice.        3.3 Route and Dose:

Subcutaneous inoculation in the prescapular region either singly or ascombined antigens into a single site. Final volume of inoculum=200 μl

-   Group A. 15 μl TRP2+85 μl PBS+100 μl TMG-   Group B. 20 μl TRP5+80 μl PBS+100 μl TMG-   Group C. 20 μl TRP2+25 μl TRP5+55 μl PBS+100 μl TMG-   Group D. 20 μl TRP2+5 μl TRP6+75 μl PBS+100 μl TMG-   Group E. 25 μl TRP5+5 μl TRP6+70 μl PBS+100 μl TMG-   Group F. 10 μl GST+90 μl PBS+100 μl TMG-   Group G. 100 μl TM+100 μl PBS Group H. untreated control group    3.4 Vaccination Scheme:

All animals were immunized on the same day.

Serum samples were collected from the half of the exp. mice from sinusorbitalis in each of the 8 groups 12 to 14 days after immunisation (30μl to 50 μl serum/animal) and then 25 days after immunisation from allremaining mice.

3.5 TBE Virus Infection and Transmission

TBE virus donors used to infect the mice were field collected Ixodesricinus female ticks (FIR) inoculated parenterally with TBE virus (Hyprstrain), diluted 10⁻¹, volume 0.002 ml, in total 5000 PFU/tick. Theywere inoculated on either the same day or the day after immunisation ofthe mice.

Recipient ticks used to assay for virus transmission were uninfectedIxodes ricinus males (MIR) and nymphs (NIR) although male ticks were nottested. Mice were challenged with 1 infected FIR, 1 MIR, and 15 NIR allplaced in one retaining chamber glued to each mouse.

No signs of inflammation were recorded during the trial.

The mice were infested with ticks either 32 days (mouse 1-5 in eachgroup+C6 and C7) or 33 days (mouse 6-10 in each group minus C6 and C7)after immunisation. Each mouse was challenged with one infected I.ricinus female+one uninfected I. ricinus male and 15 I. ricinus nymphs.Ticks fed for 3 days and were then collected. Mice were observed forsigns of illness/death for a 21 day period.

3.6 Lethal Virus Challenge

All surviving mice were inoculated intra-peritoneally with approximately1000 plaque forming units of TBE virus (Hypr strain), 54 days afterimmunisation, and then monitored for 21 days.

Survivors were bled 20 days after lethal challenge.

3.7 Results (Trial 1)

The results are summarised in Table 3 and illustrated in FIG. 6.

-   1. Highest % survival was shown by mice immunised with trp5 or trp2.-   2. Transmission-blocking, as measured by the % tick infected, was    particularly striking for trp5.-   3. The percentage of mice that support virus transmission was lowest    for trp5.    3.8 Trial II    Immunisation Protocol—Antigens Used

TRP-GST fusion proteins including: TRP2, TRP5, and GST protein(control);

Treatment Groups

Two Treatments:

-   1. TRP2 (A);    -   2. TRP5 (B);        Two Controls:-   1. GST protein (C);-   2. unimmunised (D).    Mouse Trial (Balb/c): 20 Mice per Treatment Group-   (A) 15 ul TRP2+85 ul PBS+100 ul TMG=200 ul final volume injected    S.C.-   (B) 20 ul TRP5+80 ul PBS+100 ul TMG=200 ul final volume injected    S.C.-   (C) 10 ul GST+90 ul PBS+100 ul TMG=200 ul final volume injected S.C.-   (D) unimmunised control group-   (TMG=TiterMax Gold adjuvant)    Dates for Pre-Challenge Procedures-   1. Immunisations—single dose per mouse were performed on Nov. 7,    2001, (i.e. no boostings were performed)-   2. Serum samples—were collected third week post-immunisations (Nov.    27-28, 2001) for serological assays (ELISAs) and mice were    challenged at fourth week post-immunisations (Dec. 4-7, 2001);-   TBE virus infection via Ixodes ricinus infected adult female ticks    co-feeding with uninfected I. r. nymphs was tested for    transmission/survival studies (i.e. challenge)    Results:

The results for survival of mice after commencement of infected tickfeeding (in days and date of death) and transmission rate of TBE virusare given below in Table 3: TABLE 3 Survival in D Ticks inf./fedSurvival in D Ticks inf./fed Mouse no. (death date) (% infected) Mouseno. (death date) (% infected) A1  13 (13/12) 1/11 (9.1%) B1  12 (12/12)0/14 (0%) A2 — ND B2 S; 10 (14/01)  0/3 (0%) A3  10 (10/12)  0/8 (0%) B3S; S 0/10 (0%) A4  18 (18/12)  0/8 (0%) B4  11 (11/12) 5/12 (41.7%) A5S; S 0/10 (0%) B5 S; 10 (14/01) 0/11 (0%) A6  10 (10/12)  0/8 (0%) B6 S;S  0/4 (0%) A7  10 (10/12) 2/12 (16.7%) B7  10 (10/12)  0/7 (0%) A8 S; S 3/7 (42.9%) B8 S; S  2/7 (28.6%) A9  10 (10/12) 1/10 (10%) B9 S; S  2/9(22.2%) A10 S; S 0/13 (0%) B10  12 (12/12) 1/10 (10%) A  3/9 (33%) 7/87, 8.0% B 6/10 (60%) 10/87, 11.5% C1 S; S  3/6 (50%) D1  10 (10/12) 3/7 (42.8%) C2  10 (10/12)  8/9 (88.9%) D2  12 (12/12) 7/10 (70%) C3 14 (14/12) 1/11 (9.1%) D3   6 (06/12) 4/11 (36.4%) C4  11 (11/12) 9/10(90%) D4  10 (10/12)  3/8 (37.5%) C5  11 (11/12) 3/10 (30%) D5  10(10/12)  1/2 (50%) C6  14 (14/12)  5/7 (71.4%) D6  10 (10/12)  3/6 (50%)C7  13 (13/12) 5/12 (41.7%) D7  10 (10/12) 5/11 (45.5%) C8  14 (14/12) 2/9 (22.2%) D8  10 (10/12)  3/5 (60%) C9 S; S  4/7 (57.1%) D9  10(10/12)  3/4 (75%) C10  14 (14/12)  2/6 (33.3%) D10  10 (10/12)  4/7(57.1%) C 2/10 (20%) 42/87, 48.3% D 0/10 (0%) 36/71, 50.7%Notes:ND: not done;S—surviving mouse;S—a mouse surviving 2^(nd) challenge;NEG.—donor tick was not infected, a mouse and ticks feeding on thatmouse excluded from the trial.2nd challenge control mice - challenge Hypr ip 1000 PFU (Apr. 01, 2002):1. D7, 2. D8, 3. D8, 4. D8 - mean incubation period D7.75

A summary of TBE virus transmission in the BALB/c mice is given below inTable 4. TABLE 10 Mouse Nymphs group & no. infected/fed % infected A1 1/11  9.1% A2 ND — A3 0/8   0% A4 0/8   0% A5  0/10   0% A6 0/8   0% A7 2/12 16.7% A8 3/7 42.9% A9  1/10   10% A10  0/13   0% A  7/87  8.0%(4/9) B1  0/14   0% B2 0/3   0% B3  0/10   0% B4  5/12 41.7% B5  0/11  0% B6 0/4   0% B7 0/7   0% B8 2/7 28.6% B9 2/9 22.2% B10  1/10   10% B10/87 11.5% (4/10) C1 3/6   50% C2 8/9 88.9% C3  1/11  9.1% C4  9/10  90% C5  3/10   30% C6 5/7 71.4% C7  5/12 41.7% C8 2/9 22.2% C9 4/757.1% C10 2/6 33.3% C = F 42/87 48.3% (10/10) D1 3/7 42.8% D2  7/10  70% D3  4/11 36.4% D4 3/8 37.5% D5 1/2   50% D6 3/6   50% D7  5/1145.4% D8 3/5   60% D9 3/4   75% D10 4/7 57.1 D = H 36/71 50.7% (10/10)

The combined results of TBE virus transmission on TRP immune Balb/cmice, for both Trial I & II are given below in Table 5. TABLE 5 Supportof Group of mice Transmission transmis. Survival Group A (Trp2) 14.3%(22/154)  59% (10/17) 41.2% (7/17) Group B (Trp5)  9.4% (16/180)  30%(6/20)   55% (11/20) Group C = F (Gst) 41.9% (67/160)  85% (17/20)   20%(4/20) Group D = H 58.4% (94/161) 100% (20/20)   15% (3/20)

The results of Trial II are illustrated in FIG. 7.

The combined results of Trials I and II are illustrated in FIG. 8.

3.9 Conclusions

Protection against TBEV challenges (i.e. survival of mice) was observedin the TRP2— and TRP5-immunised groups (41% and 55%, respectively)compared with much lower survival rate in control groups of unimmunisedand GST-immunized mice (15 and 20%, respectively). The infection rate inI. ricinus nymphs co-fed on mice immunised with 64TRP5 and 64TRP2 wasgreatly reduced (14% and 9%, respectively) as compared to nymphs co-fedon unimmunised and GST-immunised control animals (58% and 42%,respectively). Virus propagation (i.e. replication in host cells) wasevident in mice from both control groups while only 30% and 59% of64TRP5— and 64TRP2— immunised mice, respectively, supported TBE virustransmission.

Immunisation of Balb/c mice with tick derived recombinant 64TRP2 and64TRP5 proteins was thus shown to protect the mice against lethalchallenge with tick-infected TBE virus, as well as having a virustransmission-blocking effect, suggesting that these proteins provide aconsiderable degree of protection against virus transmission and againstvirus-induced death.

3.10 Comparison Between Efficacy of 64TRP Constructs and Commercial TBEVaccine

Experiments were performed to evaluate the efficacy of a commercial TBEvaccine and 64TRP constructs according to the invention. The data arepresented below in Table 6.

These data show that mice immunised with the commercial TBE virusvaccine are protected against lethal challenge with virus-infected ticks(although 2 mice succumbed) but are not protected against infection asshown by their ability to support virus transmission to uninfected ticksfeeding upon them.

This result contrasts with the impressive results for 64TRP5 showingmarkedly reduced levels of infection (and equally impressive protectiongiven that the immunogen is tick derived and unrelated to the virus).

It is possible that the deficiencies of the commercial TBE vaccine canbe explained by the fact that development of the commercial TBE vaccinewas based on challenging mice with virus inoculated by needle & syringeand NOT delivered by the natural route—an infected tick. It appears thatthat tick-borne delivery of the virus is able to ‘protect’ the virusfrom host immunity to a limited degree that occasionally allows thevirus to ‘breakthrough’ and cause disease/death.

This result strongly suggests that the efficacy of the constructsaccording to the invention in preventing viral transmission might becombined with the advantages of a TBE virus vaccine in protectingagainst lethal viral challenge with virus-infected ticks. Such acombined vaccine should protect against both infection and death. TABLE6 A summary of transmission efficiency of TBE virus on laboratory mice(+/− immunized with 64TRP, or a TBE virus vaccine) and their survivalafter an infected tick bite; trial I & II. Group of mice Nymphs Micesupporting Mice survived/ (immunized with) infected/fed (%) trans./used(%)* used (%) A (TRP2) 22/144 (15%) 10/16 (62%)  6/16 (38%) B (TRP5)16/173 (9%)  6/19 (32%) 10/19 (53%) F (GST + TMG) 67/153 (44%) 17/19(90%)  3/19 (16%) H (untreated) 94/161 (58%) 20/20 (100%)  3/20 (15%) I(TBE vaccine) 38/180 (21%) 12/17 (71%) 15/17 (88%)*animals supporting transmission/animals in the experiment; % of animalssupporting transmission

1. A vaccine composition effective against an infectious disease borneby a blood-feeding ectoparasite, said vaccine comprising as an activecomponent a 64p protein comprising the sequence presented in FIG. 1, afragment thereof or a homologue of said 64p protein or protein fragmentthat exhibits at least 50% sequence identity with said protein orprotein fragment.
 2. A composition according to claim 1, wherein saidprotein, fragment or homologue contains an immunogenic epitope that ispresent in a gut protein of a blood-feeding ectoparasite.
 3. Acomposition according to claim 1, wherein said infectious disease agentborne by the blood-feeding ectoparasite is the cause of Malaria; DengueFever; Yellow Fever and Arboviral Encephalitides, including EasternEquine Encephalitis, Japanese Encephalitis, LaCrosse Encephalitis, St.Louis Encephalitis, Western Equine Encephalitis, West Nile VirusEncephalitis; Lymphatic filariasis; plague; schistosomiasis;trypanosomiasis; Leishmaniasis; Onchocerciasis; protozoan, rickettsialand viral disease agents of livestock, including those causing EastCoast Fever, babesioses, anaplasmoses, cowdriosis and tick-associateddermatophilosis; and the human diseases Lyme Disease; SouthernTick-Associated Rash Illness (STARI); Babesiosis; Ehrlichiosis; RockyMountain SpottedFever; tularemia; tick-borne relapsing fever, tick-borneencephalitis and Crimean-Congo Haemorrhagic Fever.
 4. A compositionaccording to claim 1, wherein said vaccine is effective against thetransmission of agents causing disease in mammals (particularly humansand livestock), birds, reptiles and fish.
 5. A composition to claim 1,wherein said 64p fragment or homologue is derived from a blood-feedingectoparasite.
 6. A composition according to claim 5, wherein saidblood-feeding ectoparasite is a mosquito, horsefly, sandfly, blackfly,tsetse fly, flea, lice, mite, leech, flatworm or tick.
 7. A compositionaccording to claim 6, wherein said blood-feeding ectoparasite is a tick.8. A composition according to claim 1, wherein said fragment is any oneof 64trp1, 64trp2, 64trp3, 64trp4, 64trp5 or 64trp6 identified herein,or a homologue thereof.
 9. A composition according to claim 8, whereinsaid fragment comprises residues 103-132 inclusive of the sequencepresented in FIG. 1 (64trp1), or is a homologue thereof.
 10. Acomposition according to claim 8, wherein said fragment comprisesresidues 1-51 inclusive of the sequence presented in FIG. 1 (64trp2), oris a homologue thereof.
 11. A composition according to claim 8, whereinsaid fragment comprises residues 1-70 inclusive of the sequencepresented in FIG. 1 (64trp3), or is a homologue thereof.
 12. Acomposition according to claim 8, wherein said fragment comprisesresidues 69-132 inclusive of the sequence presented in FIG. 1 (64trp4),or is a homologue thereof.
 13. A composition according to claim 8,wherein said fragment comprises residues 1-133 inclusive of the sequencepresented in FIG. 1 (64trp5), or is a homologue thereof.
 14. Acomposition according to claim 1 that is effective against both adultand immature forms of the blood-feeding ectoparasite and wherein theblood-feeding ectoparasite is a tick.
 15. A composition according toclaim 1, wherein said 64p protein, fragment or homologue is expressed inrecombinant form.
 16. A composition according to claim 1, additionallycomprising a second active agent.
 17. A composition according to claim16, wherein said second active agent is a second immunogenic protein, orprotein fragment derived from a blood-feeding ectoparasite.
 18. Acomposition according to claim 17, wherein said second immunogenicprotein, fragment or homologue is a 64p protein or protein fragment. 19.A composition according to claim 18, comprising a combination of 64trp2and 64trp5; a combination of 64trp5 and 64trp6; or a combination of64trp2 and 64trp6.
 20. A composition according to claim 16, wherein saidactive agent is a vaccine against an infectious disease.
 21. Acomposition according to claim 20, wherein said vaccine against aninfectious disease is a TBE vaccine.
 22. A composition according toclaim 1, additionally comprising an adjuvant.
 23. An antibody or anantiserum that is reactive with a 64p protein, fragment or homologue.24. An antibody or antiserum according to claim 23, wherein said 64pprotein, fragment or homologue is selected from the group consisting ofthe 64p protein comprising the sequence set forth in FIG. 1, proteins,fragments, and homologues thereof.
 25. A method of production of anantibody or an antiserum, comprising immunising an animal with a vaccinecomposition in accordance with claim
 1. 26. A method of immunising ananimal against a blood-feeding ectoparasite and/or transmission of aninfectious disease caused by a blood-feeding ectoparasite comprisingadministering to said animal a vaccine composition in accordance withclaim
 1. 27. A 64p protein, fragment thereof or homologue of said 64pprotein or protein fragment, for use in a vaccine.
 28. (canceled)